Damage or loss of mechanosensory hair cells in the inner ear is a leading cause of permanent hearing loss in humans. Loss of hair cells is irreversible in mammals, but in birds and other non-mammalian vertebrates, hair cells can regenerate after being damaged by ototoxic drugs, such as the aminoglycoside neomycin. This project will use the hair cells residing in neuromasts of the lateral line system of zebrafish as a model to study regeneration after neomycin-induced hair cell death and normal hair cell turnover. The overall goal is to more fully understand the generation of hair cells and the mechanisms that guide their replacement. Preliminary data support the hypothesis that support cells within the neuromast proliferate and eventually differentiate into functional hair cells, replacing those lost during normal turnover. The synchronous hair cell loss following neomycin exposure results in upregulation of support cell proliferation, hence accelerating this process in regeneration. However, there is also evidence for a hair cell progenitor population within the neuromasts that directly differentiates into hair cells. Specific Aim 1 proposes to test the hypothesis that support cells within the neuromast undergo different, but highly stereotyped sequences of proliferative events that lead to the development of hair cell progenitors during normal hair cell addition and during regeneration. This will be achieved through a combination of in situ hybridizations for the presence and formation of hair cell progenitors, incorporation of halogenated thymidine analogs to study support cell proliferation, and live time-lapse imaging of gfp-transgenic zebrafish to follow individual cell divisions. Initial studies also implicate a role for Notch signaling in regulating these processes (Specific Aim 2). Notch inhibition studies will be performed using the gamma secretase inhibitor DAPT, and a dominant-negative Suppressor of Hairless transgenic fish under the control of a heat-shock promoter. Localization of Notch signaling components will be observed through in situ hybridization. Finally, Specific Aim 3 will determine whether the entire regeneration process is conserved in juvenile and adult fish. Hearing loss due to ototoxicity of aminoglycosides is estimated to affect 2-20% of the 2-4 million individuals treated annually. By gaining a better understanding of hair cell regeneration and its underlying mechanisms in zebrafish, we can identify genes and signaling pathways that may help in promoting the regeneration of hair cells in humans. [unreadable] [unreadable] [unreadable]